Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation

Neural progenitor cells (NPCs) have the capability to self-renew and differentiate into neurons and glial cells. In the adult brain, NPCs are found near brain microvascular networks (BMVNs) in specialized microenvironments called the neurovascular niche (NVN). Although several in vitro NVN models have been previously reported, most do not properly recapitulate the intimate cellular interactions between NPCs and perfused brain microvessels. Here, we developed perfused BMVNs composed of primary human brain endothelial cells, pericytes, and astrocytes within microfluidic devices. When induced pluripotent stem cell–derived NPCs were introduced into BMVNs, we found that NPC survival, neurogenesis, and maturation were enhanced. The application of flow during BMVN coculture was also beneficial for neuron differentiation. Collectively, our work highlighted the important role of BMVNs and flow in NPC self-renewal and neurogenesis, as well as demonstrated our model’s potential to study the biological and physical interactions of human NVN in vitro.


S1.1 Microsphere Perfusion Assay
After BMVNs were formed in MFDs using methods described in Sections 4.3 and 4.11, anastomosis was assessed using Dragon Green TM polystyrene microspheres (1.9 µm diameter, Bang Laboratories, Cat: FSDG005). Briefly, microspheres were resuspended in EGM-2 at a ratio of 1:1000. For all samples, 50 µL of unadulterated EGM-2 were added to both reservoirs on one side of the hydrogel channel and 70 µL of microsphere solution were added to the remaining two reservoirs. Immediately afterwards, fluorescence timelapse images (15 or 30 seconds with 150 millisecond intervals) were acquired using an Eclipse Ti2 microscope with a 10X objective.
Timelapse images were represented as MIPs.

S1.2 Dextran Perfusion Assay
After BMVNs were formed in MFDs using methods described in Section 4.3, microvessel permeability was assessed using fluorescent dextran. Briefly, Oregon Green TM 488 dextran (70 kDa, Invitrogen, Cat: D7173) was dissolved in EGM-2 at a concentration of 5 μg/mL. For all samples, 50 µL of unadulterated EGM-2 were added to both reservoirs on one side of the hydrogel channel and 70 µL of dextran solution were added to the remaining two reservoirs. Immediately afterwards, fluorescence timelapse images (120 seconds with 15 second intervals) were acquired using an Eclipse Ti2 microscope with a 10X objective.

S1.3 BEC Angiogenesis Assay
To characterize the angiogenic effect of NPCs and NPreCs, we investigated their influence on BEC angiogenesis in MFDs. BECs were stimulated to produce angiogenic sprouts into fibrin matrices without cells (BEC condition), with PCs (BEC-PC condition), with ACs (BEC-AC condition), with NPCs (BEC-NPC conditions), and with NPreCs (BEC-NPreC condition). Briefly, using methods similar to those in Section 4.3, PCs, ACs, NPCs, and NPreCs were resuspended separately in fibrin gels within hydrogel channels at a density of 2×10 6 cells/mL. For BEC conditions, MFDs were injected with fibrin gels containing no supporting cells. Next, BECs-tdT or BECs-EGFP were resuspended in EGM-2 at a density of 5×10 6 cells/mL. Solutions of BECs-tdT and BECs-EGFP were used in experiments to investigate the angiogenic effect of NPCs and NPreCs, respectively. For all samples, BEC solutions were added to one fluidic channel and MFDs were immediately incubated at a 90° angle for 15 minutes to promote BEC adherence to the fibrin gel. Afterwards, nonadherent BECs were washed away with PBS. Samples with BECs-tdT and BECs-EGFP were cultured in EGM-2:NPM-2 and EGM-2:NMM, respectively, under flow conditions for 7 days. On Day 1, Day 3, and Day 7, fluorescence z-stack (200 µm range with 10 µm intervals) images of angiogenic sprouts were acquired using an Eclipse Ti2 microscope with a 20X objective. In ImageJ, z-stacks were compressed to MIPs and ROIs were selected to include all angiogenic sprouts originating from between four adjacent microposts. BECs were identified by either tdTomato or EGFP signals. At each time point, the number of angiogenic sprouts in each ROI was counted. Sprout length was measured as the distance between the hydrogel-liquid interface and the tip of each sprout. This value was averaged between all sprouts in each ROI.

S1.4 BEC Vasculogenesis Assay
To characterize the vasculogenic effect of NPCs and NPreCs, we investigated their influence on BEC vasculogenesis in MFDs. BECs were stimulated to form vascular networks in fibrin gels with different supporting cells using methods similar to those in Section 4.3. Briefly, BECs-tdT or BECs-EGFP were resuspended in fibrin alone (BEC condition), with PCs (BEC-PC condition), with ACs (BEC-AC condition), with NPCs (BEC-NPC condition), and with NPreCs (BEC-NPreC condition) within hydrogel channels. Samples with BECs-tdT and BECs-EGFP were used in experiments to study NPCs and NPreCs, respectively. The final cell density of both BECs-tdT and BECs-EGFP was 6×10 6 cells/mL. The final cell density of PCs, ACs, NPCs, and NPreCs was 2×10 6 cells/mL. Samples with BECs-tdT and BECs-EGFP were cultured in EGM-2:NPM-2 and EGM-2:NMM, respectively, under flow conditions for 7 days. On Day 7, fluorescence z-stack (200 µm range with 10 µm intervals) images of vascular networks were acquired using an Eclipse Ti2 microscope with a 10X objective.

S1.5 Microvessel Analysis
To quantify and compare BMVN characteristics, microvessel analysis was performed.
Briefly, fluorescence z-stack images were acquired from samples containing BMVNs using either an Eclipse Ti2 microscope or a LSM 800 confocal microscope. Z-stacks were then converted to MIPs in ImageJ. EGFP or tdTomato signals were used to identify microvessels in images. Blood vessel area was measured as the area of microvessels within a given ROI and represented as a percentage of the total area. Using Skeletonize and Analyze Skeleton in ImageJ, average branch diameter was calculated using the following equation: where is blood vessel area, is the number of vessel branches, and is the average vessel branch length. We also measured the number of vessel segments, which was defined as the number of continuous, interconnected microvessels in each ROI.